The invention relates to an ion storage device storing ions of both polarities simultaneously for reactions between positive and negative ions, in particular for fragmentation reactions caused by electron transfer dissociation (ETD).
In the following, the term “mass” does not refer to the “physical mass” m, but to the “charge-related mass” m/z, where z is the number of excess elementary charges on the ion. The charge-related mass m/z is often (wrongly) called “mass-to-charge ratio”. Whenever reference is made simply to “mass” or to “mass of the ions”, this is always to be understood as the charge-related mass m/z, unless explicitly stated otherwise. The terms “light ions” and “heavy ions” also refer to the charge-related mass m/z.
Research into the structures, properties and activities of proteins, and also of other biopolymers, is based to a large extent on tandem mass spectrometry. Tandem mass spectrometry not only delivers spectra of the mixtures of protein ions, but also subjects individual protein ions to particular reactions, and can investigate the products of those reactions. A particularly interesting and frequently used type of such reactions is fragmentation, in which “parent ions” are first selected for fragmentation and then fragmented into daughter ions, so permitting the daughter ions created to be measured in a mass spectrum. The daughter ion mass spectra contain information about the primary and secondary structures of the proteins, enabling not only detection of the genetically determined fundamental structure of their amino acids (the “sequence”), according to type and location, but also detection of additional modifications that are important because they change the function (“post-translational modifications”, PTM).
The three individual steps of tandem mass spectrometry, (1) selection of the analyte ions to be investigated; (2) modifying reactions; and (3) analysis of the mass of the reaction products, can be carried out in storage mass spectrometers such as ion traps sequentially in the same storage unit (“tandem-in-time”). It is also possible to carry out selection of the analyte ions to be investigated in a first mass analyzer (the “mass selector”), the reactions in a special cell, and the mass analysis in a second mass analyzer (“tandem-in-space”). The invention relates to the ion-storage reaction cell in such a tandem mass spectrometer with spatially separated mass selector and mass analyzer.
Because of the high demands for a fast spectrum acquisition rate and high mass accuracy, it is particularly advantageous to measure the resulting reaction products in time-of-flight mass spectrometers with orthogonal ion injection (OTOF-MS). As a second option, modern Kingdon ion traps or ion cyclotron resonance mass spectrometers may be considered due to their high mass resolution, but only if the speed of measurement does not play the most essential role, since these Fourier transform mass spectrometers have a slow spectrum acquisition rate.
For the fragmentation of proteins or similar biopolymers, there are essentially only two fundamentally different types of fragmentation, “ergodic” and “non-ergodic” or “electron-induced” fragmentation, for both of which, however, there are a variety of versions. Ergodic fragmentation methods include collisionally induced fragmentation of ions based on multiple collisions with the molecules of a collision gas (CID=collision-induced dissociation). CID has some disadvantages, such as a limited mass range for the daughter ions, and heavy ions are hard to fragment at all. A more suitable method called “electron-induced fragmentation” is fragmentation by collisions with energetic atomic ions of opposite polarity. With respect to electron-induced fragmentation, the outstanding method is “electron transfer dissociation” (ETD), a fragmenting reaction between positive and negative ions, both of low kinetic energy.
The two fragmentation methods, ergodic and electron-induced, result in two substantially different types of fragment ion spectra. The information they contain is complementary, and measuring both kinds of fragment ion spectra leads to particularly in-depth information about the structures of the analyte ions. As is known to those skilled in the art, the fragment ions from electron-induced fragmentation belong to the c and z series of fragmentation, and are therefore very different from the fragment ions of the b and y series that are obtained from ergodic fragmentation. In particular, however, electron-transfer dissociation retains almost all the side chains that are lost in ergodic fragmentation, including the important post-translational modifications such as phosphorylations, sulfations and glycosylations. A comparison of good quality fragmentation ion spectra obtained from ergodic and electron-induced fragmentation thus exhibits presence and location of post-translational modifications. The comparison is also advantageous, or even essential, for other investigations such as de novo sequencing.
This invention relates particularly to a suitable reaction cell for electron transfer dissociation. It is much to be preferred if both ergodic fragmentation—such as collisionally induced fragmentation (CID)—and electron-transfer dissociation (ETD) could be performed in the same reaction cell. Both types of fragmentation ion spectra should meet the highest quality demands. A modern tandem mass spectrometer for biological analysis must offer fully effective methods for both types of fragmentation.
Electron transfer dissociation can easily be carried out in ion traps in which both positive and negative ions can be stored and react with one another, by introducing suitable negative ions to the stored positive analyte ions. Methods of this type are described in the patent publications US 2005/0199804 A1 (D. F. Hunt et al.) and DE 10 2005 004 324.0 (R. Hartmer and A. Brekenfeld).
The fragmentation of protein ions by electron transfer is generated by reactions between multiply-charged positive protein ions and suitable negative reactant ions. Suitable negative reactant ions are usually specially selected radical anions, such as those of fluoranthene, fluorenone, anthracene or other polyaromatic compounds. Some monoaromatic or even non-aromatic compounds, e.g. 1-3-5-7-Cyclooctatetraen, may be used, too. These radical anions can very easily donate electrons to form stable, neutral molecules with complete electron configuration. They are generated, as described in the two patent applications quoted above, in NCI ion sources (NCI=“negative chemical ionization”) by simple electron capture or by electron transfer. NCI ion sources have essentially the same design as chemical ionization (CI) ion sources, but they are operated in a different way in order to obtain large quantities of low-energy electrons. NCI ion sources are also referred to as electron attachment ion sources.
The radical anions of suitable substances can, however, also be generated directly or indirectly in electrospray ion sources, generally used in time-of-flight mass spectrometers with orthogonal ion injection. Indirect generation means that anions of certain substances are first generated, and these are then converted by careful collisionally induced fragmentation into the radical anions that can be used as reactant anions for ETD (see, e.g. “Electron-Transfer Reagent Anion Formation via Electrospray Ionization and Collision-Induced Dissociation”, T.-Y. Huang et al., Anal. Chem. 2006, 78, 7387-7391).
Up to now, exclusively linear ion traps (“2D ion traps”) have been used as separate ETD fragmentation cells in tandem mass spectrometers with high-resolution mass analyzers. Although ETD fragmentation can also be carried out in three-dimensional ion traps (“3D ion traps”), commercial 3D ion traps used in this way are restricted exclusively to those mass spectrometers that use these 3D ion traps simultaneously as mass analyzers for measurement of the fragment ion spectra. They are not intended to transfer the fragment ions into another mass analyzer, and this is only possible with some difficulty and expense.
In linear ion traps, the freshly introduced parent ions are stored, after their kinetic energy has been damped by the collision gas, in the form of a thread-like cloud of small diameter along the longitudinal axis of the rod system. For fragmentation by electron transfer, parent ions that have at least two, but preferably three, four, five or more charges, are selected; in extreme cases, parent ions with 10 or even 15 charges are fragmented.
Linear ion traps are generally designed as multipole rod systems, as quadrupole, hexapole or octopole rod systems having two, three or four pairs of pole rods. A hexapole rod system is illustrated in FIG. 1. The two opposite phases of an RF voltage are applied alternately around the pole rods, generating a radially repelling pseudopotential inside. Quadrupole rod systems exhibit a quadratic rise in the pseudopotential in a radial direction, and the radial oscillations of the (undamped) ions are harmonic. Under the influence of the damping gas, they accumulate as a thread-like cloud along the axis of the rod system. Hexapole rod systems are most often used as the collision cells for fragmentation; they exhibit a cubic rise in the pseudopotential, and the pseudopotential well across the axis therefore has a flatter bottom. Due to the lower repelling force near the axis, the thread-like cloud has a somewhat larger diameter.
In this document, all systems that radially confine ions, including multipole rod systems in particular, are referred to as “ion guide systems”, since ions can be canalized inside. In this sense, linear ion traps, quadrupole mass filters, and so-called ion funnels, are examples of ion guide systems, even if their primary purpose is different.
A “pseudopotential” is not a real potential, but describes the time-averaged effects of the force exerted by an inhomogeneous RF field on ions of both polarities. An RF voltage that is present at the tip of an electrode, a wire, or indeed on a pole rod, creates an inhomogeneous electrical field of this sort, and thus a pseudopotential, driving ions away from the tip or wire. An RF dipole field also generates a pseudopotential that repels ions away from the dipole, only ions exactly on the axis of the dipole are driven on a highly instable path toward the center of the dipole.
Although methods are being developed for bringing analyte ions into reaction with a continuous flow of reactant ions, it has so far appeared appropriate to confine both types of ions simultaneously in a reaction cell, so that the reactions between the positive and negative ions can proceed in an undisturbed, controlled manner. The basic principle used for this storage has been known for a long time. US patent specification U.S. Pat. No. 5,572,035 A (“Method and Device for the Reflection of Charged Particles on Surfaces”; J. Franzen 1995) already comments that: “All types of cylindrical or conical ion guides . . . can be used as storage devices if the end openings are barred for the exit of ions by reflecting rf or dc potentials. With rf field reflection, ions of both polarities can be stored. With dc potentials, ion guides store ions of a single polarity only.” (The underlining has been added). This patent specification is concerned in a very general way with the reflection of ions of both polarities at pseudopotentials formed by inhomogeneous RF fields.
The confinement of ions in linear ion traps, which constitute ion guides as defined in this document, using RF-generated pseudopotential barriers has thus been known for a long time. There are, however, a range of different implementations. The review article by Y. Xia and S. A. McLuckey: “Evolution of Instrumentation for the Study of Gas-Phase Ion/Ion Chemistry via Mass Spectrometry”, J Am Soc Mass Spectrom 2008, 19, 173-189, provides an overview.
One embodiment is described in US patent specification 7,026,613 B2 (J. Syka, 2004), which states: “Periodic voltages are applied to electrodes in the first set of electrodes to generate a first oscillating electric potential that radially confines the ions in the ion channel, and periodic voltages are applied to electrodes in the second set of electrodes to generate a second oscillating electric potential that axially confines the ions in the ion channel”. This complicated description says in effect that a radially confining ion guide system (the first set of electrodes) is terminated by setting up a pseudopotential barrier in an axial direction with the aid of RF voltages on a second set of electrodes (necessarily positioned separately in the axial direction) for ions of both polarities. The heart of this invention is therefore simply the addition of electrodes for the axially terminating RF voltage, which, although not explicitly mentioned in the above quotation from the US patent of J. Franzen, are of course inherently necessary.
It must be explicitly pointed out that the two RF voltages in the invention of J. Syka are applied to two different sets of electrodes, both according to the description in the disclosure and according to the claims. This leads, however, to pseudopotential barriers with an unfavorable form. If the second RF voltage is applied to terminating electrodes at the end of the pole rods, for instance to apertured diaphragms, a pseudopotential with two maxima is generated. An apertured diaphragm acts like the ring electrode of a three-dimensional ion trap, and generates a storage region in the form of a pseudopotential well in the plane of the apertured diaphragm. The potential well of the storage region is terminated at both ends by a pseudopotential barrier. If these double barriers are switched on by the RF voltage at the apertured diaphragm, filling becomes difficult, since a proportion of the ions always remains in the potential well of the apertured diaphragm's storage region. For this reason, ion traps according to the prior art are always filled with the pseudopotential barriers switched off, but the resulting problems necessitate differently designed ion traps.
The only commercial device currently available in which ETD is carried out in a linear ion trap therefore operates with an ion trap that is divided into three segments. The axial potentials of these segments can be adjusted separately. This makes it possible to introduce positive and negative ions in sequence, and to hold them temporarily in different segments of the linear ion trap before equalizing the axial potentials to mix the ions and thereby initiate the reactions.
In patent specification U.S. Pat. No. 7,227,130 B2 “Method for Providing Barrier Fields at the Entrance and Exit End of a Mass Spectrometer” (J. W. Hager and F. A. Londry, 2005) auxiliary RF voltages are applied to terminating electrodes at the entrance and exit ends of a linear ion trap with long pole rods in order to confine ions of both polarities in the axial direction; the auxiliary RF voltages are obtained by means of voltage dividers from the main RF voltage at the pole rods. This is a special embodiment of J. Syka's invention.
Patent specification U.S. Pat. No. 7,288,761 B2 (B. A. Collings, 2005) describes for the first time the possibility of not applying the axially confining RF voltage to electrodes at the end of a multipole rod system, but instead making the axial potential of the rod system oscillate at high frequency with respect to the surrounding potential. In this way, axially acting pseudopotential barriers are created at the ends of the multipole rod system. Only one RF generator is required for this method. In the patent specification, the oscillating axial potential is generated either by an asymmetrical arrangement of the pole rods around the axis, or by means of two different amplitudes for at least one of the two phases of the RF voltage at the pole rods. In spite of the advantageous use of only a single RF generator, a disadvantage here is that the amplitude of the oscillating axis potential cannot easily be adapted to the mass range of the ions that are to be confined, since in order to do this either the spacing between the pole rods or the transformer for generating the two amplitudes has to be changed for at least one of the two phases of the RF voltage. In the latter case, moreover, more vacuum feedthroughs than usual have to be used, since at least two pole rods must now be supplied with voltages individually rather than in pairs. The thread-like cloud of ions no longer collects along the axis of the pole rod system. In the first case, where asymmetrically arranged pole rods are used, the axially acting pseudopotential barrier cannot be switched off at all. Also in the second case, where asymmetric amplitudes of RF voltage are used, it is difficult to switch the barrier off, since the adjustment of the resonant circuit for resonance and high Q factor is disturbed when switching to symmetrical amplitudes.